Methods of Producing 6-Carbon Monomers From 8-Carbon Compounds

Abstract

This document describes biochemical pathways for producing 6-hydroxyhexanoic acid using a monooxygenase to form a 7-hydroxyoctanoate intermediate, which can be converted to 6-hydroxyhexanoate using a polypeptide having monooxygenase, secondary alcohol dehydrogenase, or esterase activity. 6-hydroxyhexanoic acid can be enzymatically converted to adipic acid, caprolactam, 6-aminohexanoic acid, hexamethylenediamine or 1,6-hexanediol. This document also describes recombinant hosts producing 6-hydroxyhexanoic acid as well as adipic acid, caprolactam, 6-aminohexanoic acid, hexamethylenediamine and 1,6-hexanediol.

1 . A method of producing 7-hydroxyoctanoate, said method comprising enzymatically converting octanoate to 7-hydroxyoctanoate using a monooxygenase classified under EC. 1.14.14.1. 
     
     
         2 . The method of  claim 1 , wherein said monooxygenase classified under EC 1.14.14.1 has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. 
     
     
         3 . The method of  claim 1 , further comprising enzymatically converting 7-hydroxyoctanoate to 6-hydroxyhexanoate using a secondary alcohol dehydrogenase, a monooxygenase classified under EC 1.14.13.-, and an esterase. 
     
     
         4 . The method of  claim 3 , wherein said esterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 or is any other esterase classified under EC 3.1.1.1 or EC 3.1.1.3. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein octanoate is produced:
 (a) using a thioesterase having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 1, 15, 16, or 17, or any other thioesterase, to convert octanoyl-[acp] or octanoyl-CoA to octanoate; or   (b) from 2-oxononanoate using an aldehyde dehydrogenase and a decarboxylase having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 23, or any other decarboxylase.   
     
     
         7 - 9 . (canceled) 
     
     
         10 . The method of  claim 3 , wherein said secondary alcohol dehydrogenase has at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 19. 
     
     
         11 . The method of  claim 3 , wherein said monooxygenase classified under EC 1.14.13.- has at least 70% identity to the amino acid sequence set forth in SEQ ID NO:20 or SEQ ID NO: 21. 
     
     
         12 . A method for biosynthesizing 6-hydroxyhexanoate, said method comprising either:
 (i) enzymatically synthesizing 7-hydroxyoctanoate from octanoyl-CoA or octanoyl-[acp] using a thioesterase and a monooxygenase classified under EC 1.14.14.1, and enzymatically converting 7-hydroxyoctanoate to 6-hydroxyhexanoate using a secondary alcohol dehydrogenase, a monooxygenase classified under EC 1.14.13.-, and an esterase; or   (ii) enzymatically synthesizing 7-hydroxyoctanoate from 2-oxononanoate using a decarboxylase, an aldehyde dehydrogenase, and a monooxygenase classified under EC 1.14.14.1, and enzymatically converting 7-hydroxyoctanoate to 6-hydroxyhexanoate using a secondary alcohol dehydrogenase, a monooxygenase classified under EC 1.14.13.-, and an esterase;   wherein said monooxygenase classified under EC 1.14.14.1 has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18, or is any other monooxygenase classified under EC 1.14.14.1;   wherein said thioesterase has at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 1, 15, 16, or 17, or is any other thioesterase;   wherein said esterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 or is any other esterase;   where said monooxygenase classified under EC 1.14.13.- has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, or is any other monooxygenase classified under EC 1.14.13;   wherein said secondary alcohol dehydrogenase has at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 19, or is any other secondary alcohol dehydrogenase; and/or   wherein said decarboxylase has at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 23, or is any other decarboxylase.   
     
     
         13 - 19 . (canceled) 
     
     
         20 . The method of  claim 12 , said method further comprising enzymatically converting 6-hydroxyhexanoate to adipic acid, 6-aminohexanoate, caprolactam, hexamethylenediamine, or 1,6-hexanediol in one or more steps. 
     
     
         21 . The method of  claim 20 , wherein:
 (a) 6-hydroxyhexanoate is converted to adipic acid using one or more of a monooxygenase, a primary alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase;   (b) 6-hydroxyhexanoate is converted to 6-aminohexanoate using one or more of a primary alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and a ω-transaminase;   (c) 6-hydroxyhexanoate is converted to caprolactam using one or more of a primary alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a ω-transaminase, and an amidohydrolase;   (d) 6-hydroxyhexanoate is converted to hexamethylenediamine using one or more of a carboxylate reductase, a ω-transaminase, a primary alcohol dehydrogenase, an N-acetyltransferase, and an acetylputrescine deacylase; and/or   (e) 6-hydroxyhexanoate is converted to 1,6-hexanediol using a carboxylate reductase and an alcohol dehydrogenase.   
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 21 , further comprising converting 6-aminohexanoate to hexamethylenediamine using one or more of a carboxylate reductase and a ω-transaminase. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The method of  claim 21  or  23 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs. 7-12. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 20  or  23 , wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs 2-6. 
     
     
         29 . The method of  claim 1  or  claim 12 , wherein said method is performed in a recombinant host; 
     
     
         30 . The method of  claim 29 , wherein:
 (a) said host is subjected to a non-cyclical cultivation strategy to achieve aerobic, anaerobic or, micro-aerobic cultivation conditions;   (b) a cyclical cultivation strategy is used to alternate between anaerobic and aerobic cultivation conditions;   (c) said host is cultured under conditions of nutrient limitation;   (d) said host is retained using a ceramic hollow fiber membrane;   (e) the principal carbon source fed to the fermentation derives from a biological feedstock; and/or   (f) the principal carbon source fed to the fermentation derives from a non-biological feedstock.   
     
     
         31 - 34 . (canceled) 
     
     
         35 . The method of  claim 30 , wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 30 , wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO 2 /H 2 , methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams. 
     
     
         38 . The method of  claim 29 , wherein the host is a prokaryote or a eukaryote. 
     
     
         39 . The method of  claim 38 , wherein said prokaryote is from a genus selected from the group consisting of  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus , and  Rhodococcus.    
     
     
         40 . The method of  claim 39 , wherein said prokaryote is selected from the group consisting of  Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis , and  Rhodococcus equi.    
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 42 , wherein said eukaryote is selected from the group consisting of  Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issatchenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans , and  Kluyveromyces lactis.    
     
     
         44 . The method of  claim 29 , wherein:
 (a) the host's tolerance to high concentrations of a C6 building block is improved through continuous cultivation in a selective environment;   (b) said host comprises an attenuation of one or more of the following enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoacid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C6 building blocks and central precursors as substrates, a butaryl-CoA dehydrogenase, or an adipyl-CoA synthetase; and/or   (c) said host overexpresses one or more genes encoding: an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase, a transketolase, a puridine nucleotide transhydrogenase, a glyceraldehyde-3P-dehydrogenase, a malic enzyme, a glucose-6-phosphate dehydrogenase, a glucose dehydrogenase, a fructose 1,6 diphosphatase, a L-alanine dehydrogenase, a L-glutamate dehydrogenase, a formate dehydrogenase, a L-glutamine synthetase, a specific glutarate CoA-ligase, a specific 5-hydroxypentanoate dehydrogenase, a specific 5-oxopentanoate dehydrogenase, a propanoate CoA-ligase, a diamine transporter, a dicarboxylate transporter, and/or a multidrug transporter.   
     
     
         45 - 46 . (canceled) 
     
     
         47 . A recombinant host comprising at least one exogenous nucleic acid encoding (i) a monooxygenase classified under EC 1.14.14.1; (ii) a thioesterase, or a decarboxylase and an aldehyde dehydrogenase, (iii) a secondary alcohol dehydrogenase, (iv) a monooxygenase classified under EC 1.14.13.-, and (v) an esterase, said host producing 6-hydroxyhexanoate. 
     
     
         48 . The recombinant host of  claim 47 , wherein:
 (a) said monooxygenase classified under EC 1.14.14.1 has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18;   (b) said host comprising said thioesterase, said thioesterase having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 1, 15, 16, or 17;   (c) said host comprising said decarboxylase and said aldehyde dehydrogenase, said decarboxylase having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 23;   (d) said monooxygenase classified under EC 1.14.13.- has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO:21;   (e) said esterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22; and/or   (f) said secondary alcohol dehydrogenase has at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 19.   
     
     
         49 - 53 . (canceled) 
     
     
         54 . The recombinant host of  claim 47 , said host further comprising either:
 (a) one or more of the following exogenous enzymes: a monooxygenase, an alcohol dehydrogenase, a 5-oxovalerate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or an aldehyde dehydrogenase, said host further producing adipic acid;   (b) one or more of the following exogenous enzymes: a transaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, or a primary alcohol dehydrogenase, said host further producing 6-aminohexanoate;   (c) an exogenous amidohydrolase, said host further producing caprolactam;   (d) one or more of the following exogenous enzymes: a carboxylate reductase, a ω-transaminase, a deacylase, a N-acetyl transferase, or a primary alcohol dehydrogenase, said host further producing hexamethylenediamine; and/or   (e) an exogenous carboxylate reductase and an exogenous primary alcohol dehydrogenase, said host further producing 1,6-hexanediol.   
     
     
         55 - 58 . (canceled) 
     
     
         59 . A bio-derived product, bio-based product or fermentation-derived product, wherein said product comprises:
 i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound produced according to  claim 1  or  claim 12 , or any one of  FIGS. 1-5 , or any combination thereof,   ii. a bio-derived, bio-based or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of i., or any combination thereof,   iii. a bio-derived, bio-based or fermentation-derived resin comprising the bio-derived, bio-based or fermentation-derived compound or bio-derived, bio-based or fermentation-derived composition of i. or any combination thereof or the bio-derived, bio-based or fermentation-derived polymer of ii. or any combination thereof,   iv. a molded substance obtained by molding the bio-derived, bio-based or fermentation-derived polymer of ii. or the bio-derived, bio-based or fermentation-derived resin of iii., or any combination thereof,   v. a bio-derived, bio-based or fermentation-derived formulation comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., or bio-derived, bio-based or fermentation-derived molded substance of iv, or any combination thereof, or   vi. a bio-derived, bio-based or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i. bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., bio-derived, bio-based or fermentation-derived formulation of v., or bio-derived, bio-based or fermentation-derived molded substance of iv., or any combination thereof.   
     
     
         60 . A non-naturally occurring organism comprising at least one exogenous nucleic acid encoding at least one polypeptide having the activity of at least one enzyme depicted in any one of  FIGS. 1 to 5 . 
     
     
         61 . A non-naturally occurring biochemical network comprising one or more polypeptides having the activity of a monooxygenase, a secondary alcohol dehydrogenase, and an esterase A. 
     
     
         62 . A nucleic acid construct or expression vector comprising
 (a) a polynucleotide encoding a polypeptide having monooxygenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having monooxygenase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 18;   (b) a polynucleotide encoding a polypeptide having esterase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having esterase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 22;   (c) a polynucleotide encoding a polypeptide having thioesterase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having thioesterase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 1, 15, 16, or 17;   (d) a polynucleotide encoding a polypeptide having decarboxylase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having decarboxylase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 23;   (e) a polynucleotide encoding a polypeptide having alcohol dehydrogenase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having alcohol dehydrogenase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 21;   (f) a polynucleotide encoding a polypeptide having ω-transaminase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω-transaminase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 7-12;   (g) a polynucleotide encoding a polypeptide having carboxylate reductase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having carboxylate reductase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 2-6 or 24; or   (h) a polynucleotide encoding a polypeptide having monooxygenase, primary alcohol dehydrogenase, 6-hydroxyhexanoate dehydrogenase, 7-oxoheptanoate dehydrogenase, 6-oxohexanoate dehydrogenase, 5-oxovalerate dehydrogenase, aldehyde dehydrogenase, 5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxylate reductase, N-acetyltransferase, acetylputrescine deacylase or ω-transaminase activity.   
     
     
         63 . A composition comprising the nucleic acid construct or expression vector of  claim 62 .